霍氏肠杆菌BG200的分离鉴定及其镉耐受性和对镉胁迫下水稻生长的影响

Isolation and identification of Enterobacter hormaechei BG200 with cadmium tolerance and promoting effect on rice growth under cadmium stress

摘要:

【背景】生物性环境修复和保护策略是发展可持续农业、保障粮食生产、保证食品质量安全的重要策略，因此挖掘并解析对环境镉污染有修复和保护潜能的菌株对农业生产具有重要意义。【目的】揭示霍氏肠杆菌(Enterobacter hormaechei) BG200耐镉性能及其机制，探究霍氏肠杆菌BG200对镉胁迫下水稻幼苗生长性能的影响。【方法】以低镉环境中富镉的灰褐牛肝菌(Boletus griseus)子实体为原料，采用含镉平板培养法对其内生细菌进行分离、纯化并进行16S rRNA基因扩增和测序；采用含镉摇瓶明确菌株的镉耐受性及其对镉的去除作用；利用转录组测序技术和生物信息学分析菌株在镉胁迫下差异表达基因；采用水培实验考察耐镉菌液对镉胁迫下水稻种子发芽和幼苗生长发育的影响以及镉积累变化。【结果】分离得到一株具有镉耐性的霍氏肠杆菌BG200，镉离子(Cd²⁺)对菌株BG200生长最低抑制浓度为200 mg/L。菌株BG200对液体中Cd²⁺的吸附去除率可达69.22%。菌株BG200在Cd²⁺胁迫环境中由长杆状变为短棒状并聚集，随镉浓度增加，菌体表面镉元素占比增加；Cd²⁺可能借助ATP结合盒(ATP-binding cassette,ABC)家族和主要异化子超家族(major facilitator superfamily,MFS)转运体或锌铁离子通道进入到菌体内；菌体显著上调产能和抗氧化系统相关的基因；菌体内多重耐药转录因子MarR、超氧化物反应转录因子SoxS、外排泵MerR家族转录因子等表达上调；同时，菌体上调外排转运体蛋白细菌抗菌化合物外排家族(proteobacterial antimicrobial compound efflux family,PACE)和属于MFS外排转运蛋白的GlpT。添加BG200菌液显著提高了镉胁迫下水稻种子的萌发率，提高了水稻苗的根长、株高、叶绿素、可溶性蛋白含量、抗氧化酶活性[过氧化氢酶(catalase,CAT)、过氧化物酶(peroxidase,POD)、超氧化物歧化酶(superoxide dismutase,SOD)]；降低水稻叶片中过氧化氢(hydrogen peroxide,H₂O₂)、超氧阴离子(superoxide anion radical,O^2–)、丙二醛(malondialdehyde,MDA)和脯氨酸(proline,Pro)含量，降低水稻植株总镉含量、镉迁移系数和营养液中镉残留量。【结论】霍氏肠杆菌BG200具有较高的镉耐性和镉吸附能力，对镉胁迫条件下水稻苗有一定促生性，能降低水稻植株镉含量，在环境镉污染的微生物治理策略上具有应用潜力。

Abstract:

[Background] Environmental remediation and protection using microbial techniques is an important strategy for developing sustainable agriculture, guaranteeing food production, and ensuring food quality and safety. Therefore, it is of great significance for agricultural production to mine the strains with the potential of remediating and protecting against cadmium pollution. [Objective] To reveal the cadmium tolerance and mechanism of Enterobacter hormaechei BG200 and investigate the effect of E. hormaechei BG200 on the growth of rice seedlings under cadmium stress. [Methods] The endophytic bacteria were isolated from Boletus griseus by the cadmium-rich plate culture method. The 16S rRNA gene was amplified and sequenced for the isolated strain. The cadmium tolerance of the strain and its removal effect on cadmium were determined. Transcriptome sequencing and bioinformatics were employed to mine the differentially expressed genes of the strain under cadmium stress. Hydroponic experiments were conducted to investigate the effects of the strain on seed germination, seedling growth, and cadmium accumulation of rice under cadmium stress. [Results] E. hormaechei BG200 with cadmium tolerance was isolated from B. griseus. The minimum inhibitory concentration of cadmium ion (Cd²⁺) on the growth of E. hormaechei BG200 was 200 mg/L. The removal rate of strain BG200 for Cd²⁺ in liquid by adsorption reached 69.22%. The cells of strain BG200 exposed to Cd²⁺ stress changed from long rod to short rod. With the increase in Cd²⁺concentration, the proportion of cadmium element on the cell surface of strain BG200 increased. Cd²⁺ might enter bacterial cells via transporters of the ATP-binding cassette family and major facilitator superfamily (MFS) or zinc-iron ion channels. Under cadmium stress, strain BG200 significantly upregulated the expression of genes related to productivity and antioxidant system. In addition, the expression levels of MarR family transcription factors associated with multi-drug resistance, SoxS family transcription factors associated with superoxide reaction, and MerR family transcription factors associated with efflux pumps were upregulated in bacteria. At the same time, strain BG200 upregulated the expression of proteobacterial antimicrobial compound efflux (PACE) transporters and GlpT, an MFS member. The fermentation broth of strain BG200 significantly increased the germination rate of rice seeds under cadmium stress, and it increased the root length, plant height, chlorophyll content, soluble protein, and activities of antioxidant enzymes (catalase, peroxidase, and superoxide dismutase) of rice seedlings. Meanwhile, the fermentation broth reduced the content of hydrogen peroxide (H₂O₂), superoxide anion radical (O^2–), malondialdehyde (MDA), and proline (Pro) in rice leaves as well as the total cadmium content and cadmium migration index of rice seedlings under cadmium stress. Moreover, it decreased the cadmium residue in the nutrient solution. [Conclusion] E. hormaechei BG200 has high cadmium tolerance and adsorption capacity. The fermentation broth of strain BG200 could not only promote the growth of rice seedlings but also reduce the content of cadmium in rice seedlings under cadmium stress. E. hormaechei BG200 showcases the potential of application in the microbial remediation of environmental cadmium pollution.